HIV-Associated Neurocognitive Disorders (HAND) continue to affect ~50% of HIV(+) patients1-4, despite suppression of HIV replication with antiretroviral compounds1-4. With antiretroviral therapy, HAND is now a chronic, progressing disease leading to a spectrum of neurologic disorders. In more severe forms of disease, such as HIV-Associated Dementia (HAD), there is neuronal loss, but in milder forms of disease, synaptic damage is the more salient pathologic change8-14. Both neuronal loss and synaptic simplification are associated with macrophage infiltration and neuroinflammation 15-18, which are believed to be mediated by release of excitotoxins, reactive oxygen species (ROS), cytokines and viral proteins10,15,19-42. Transcriptomic, Proteomic, and Metabolomic approaches have identified several classes of genes and proteins that are dysregulated in HAND, including those regulating innate immunity, inflammasome formation, cell cycle regulation, myelination and synaptic development and function43-51. As synaptic damage correlates best with HAND progression, we are most interested in understanding these changes; however, the mechanisms underlying synaptodendritic damage remain elusive. A particular hurdle to understanding the contribution of gene expression in neurons is the presence of polyribosome tracts in the distal axons and dendrites of neurons where translation is regulated locally to provide rapid changes in gene expression resulting in synaptic plasticity. Local translation, splicing, and stability of RNA as wll as delivery of RNA to these sites require RNA binding proteins (RBPs). It is now clear that RNA regulation by RBPs contributes significantly to regulation of synapse formation, maintenance, and turnover as evidenced by the growing number of genetic disorders of the nervous system with defects in RBPs including Fragile X Syndrome, Frontotemporal Dementia, Spinocerabellar Ataxia, Spinomuscular Atrophy, and Parkinson disease53-55. In order to gain insight into the synaptic changes observed in neurons responding to HIV-associated neurotoxins in HAND, we propose to use a novel, high yield approach, protein interaction profile-sequencing (PIP-Seq), to identify global changes in RNA sequences bound by RBPs in neurons exposed to HIV-associated neurotoxins in an in vitro model of disease, and in the CNS of primates in an in vivo model of lentiviral encephalitis. PIP-Seq employs a novel RNase-mediated protein footprinting strategy in which all unbound RNA is digested by RNAses and RNA sequences protected by RBPs are subjected to deep sequencing. Combining information gained from PIP-seq with bioinformatics approaches, this high risk, high yield approach will provide insights into: the RBP-bound RNA sequences in neurons at baseline, the changes in RBP-bound RNA sequences in neurons responding to HIV-associated neurotoxins, the region of RNA molecules bound by RBP, the conservation of the bound sites across vertebrate species, the overrepresented biologic processes in which differentially RBP-bound RNAs function, the ability to distinguish putative SNPs linked to changes in RBP activity, and the means to identify known and novel RBPs that regulate RNAs linked to HIV-associated changes. Finally, identified RBP-bound RNA sequences will be compared with previous transcriptomic and proteomic results to gain insight into the mechanisms underlying changes in observed gene expression in HAND. This novel, high risk, high yield approach stands to provide valuable insight into changes in neuronal responses in HAND and in diseases beyond, and will provide the underpinnings for studies to increase both our understanding of basic neuronal function and how disease processes impact that function.